Extended Mass Range Triple
Quadrupole for Routine Analysis of
Authors
Linfeng Wu, Christine A. Miller,
Jordy Hsiao, Te-wei Chu,
Behrooz Zekavat, and Anabel Fandino
High Mass-to-charge Peptide Ions
Application Note
Targeted Proteomics
Introduction
Routine, high-throughput protein quantitation by LC/Triple Quadrupole analysis uses
peptides as surrogates for the corresponding proteins. A critical step to successful
analysis is to select unique peptides and suitable MRM transitions for each targeted
peptide. Peptides with high m/z precursor or product ions are often not selected for
analysis due to instrument mass range limitations. However, to address biological
questions these peptides may provide critical information or be the only analytical
choice. Some examples where this may arise are membrane proteins with large
hydrophobic transmembrane domains, proteins with extensive post-translational
modifications, and endogenously produced peptides.
This Application Note demonstrates the performance of the Agilent 6495B Triple
Quadrupole for peptides with high m/z product ions, showing the benefits of having a
mass range up to m/z 3,000. Tryptically digested monoclonal antibody (mAb) standard
and a HeLa cell membrane-enriched extract were used as simple and complex samples,
respectively.
Experimental Method
Protein/peptide selection
Using an Agilent 6550 Q-TOF Mass
Spectrometer, LC/MS data were
acquired in a data‑dependent mode
on anti-IL8 mAb digest and a HeLa
membrane‑enriched protein digest.
Raw data were searched against the
appropriate database using Agilent
Spectrum Mill (Rev B.05.00.181 SP1) to
identify peptides producing high m/z
product ions for testing the performance
of the Agilent 6495B Triple Quadrupole
Mass Spectrometer.
LC/MS/MS system
An Agilent 1290 Infinity UHPLC system
was interfaced to a 6495B Triple
Quadrupole with a mass range of 3,000 u.
The LC/MS experiments were performed
using an Agilent Jet Stream (AJS)
source, and an Agilent Poroshell 120
EC‑C18, 2.1 × 100 mm 2.7 µm column
(p/n 695775‑902). For the mAb digest,
a 3.5 minute MRM method was used
and for the HeLa membrane digest, a
21 minute MRM method was used.
Table 1. Agilent 6495 Triple Quadrupole MS method.
For glycopeptide quantification, the mAb
digest was diluted in series, and the
signal response for each injection level
was plotted against the total mAb digest
amount on-column.
For the HeLa membrane peptide
quantification, the signal response for
each product ion was plotted against the
relative injection amount on-column.

Parameter Setting
Ion mode AJS, Positive
Gas temperature 150 °C
Drying gas flow 15 L/min
Nebulizer gas 30 psi
Sheath gas temperature 200 °C
Sheath gas flow 11 L/min
Capillary voltage 3,500 V
Nozzle voltage 0V
High/Low pressure RF voltage 200/110 V
Delta EMV 100–200 V
Q1 and Q3 resolution Wide/Unit
Fragmentor 380 V
Cell accelerator voltage 4V
Cycle time 267–369 ms

Table 2. Selected peptides and ions for LC/MS/MS test.

m/z m/z MRM m/z MRM

Protein name Peptide Modification Precursor Quantifier Qualifier
Anti-IL8 mAb antibody EEQYN[+1606.6]STYR N-glycan G1F 932.7 204.1 366.1
1,392.6
2,065.8
2,227.9
Sterol O-acyltransferase 1 SSTVPIPTVNQYLYFLFAPTLIYR none 1,402.3 1,215.2 762.5
1,110.1
2,219.2
2,429.3
ATP Synthase subunit d, NLIPFDQMTIEDLNEAFPETK none 1,233.1 1,063.0 228.1
mitochondrial 341.2
474.3
2,125.0
ADP/ATP Translocase 1 YFAGNLASGGAAGATSLC[+57FVYPLDFAR Carbamidomethylation 1,398.7 718.4 175.1
881.5
1,915.9
2,060.0

2
 Results and Discussion Figure 1 and Figure 2 show the MS/MS Product ions with higher m/z tend to
spectrum and the optimized MRM have less background noise or higher
Peptide identification and selection chromatography for two of the peptides. signal-to-noise ratios in complex samples.
Anti-IL8 mAb digest and HeLa membrane The product ions selected for the MRM
digest were used for peptide identification method are marked with * in the MS/MS
and MRM method development with the plot.
following results:
Glycopeptides produce high m/z product
One G1F N-glycopeptide from the mAb ions, which have large glycan chains.
digest, and three peptides from the HeLa These product ions are informative for
membrane digest with product ions interpreting glycan structure.
ranging from m/z 204.1 to m/z 2,429.3
were selected for a LC/MS/MS test
(Table 2).

B
×10 3 ×10 4
A 1

Counts
9
0
8
×10 3
7
6 Counts 0
×10 2
Counts

5
Counts

4 0
×10 2
3
Counts

1
2
×10 2
1
Counts

0
200 400 600 800 1,000 1,200 1,400 1,600 1,800 2,000 2,200 0.9 1.0 1.1
Acquisition time (min)
Mass-to-charge (m/z)

Figure 1. MS/MS spectrum and the optimized MRM chromatography for G1F glycopeptide EEQYN[+1606.6]STYR.

precursor
A B
×103 ×10 2
Counts

y18 2
3.2
y20
2.8 y20+2 ×10 2
Counts

2
2.4 y6 y18+2
2.0 ×10 2
5
Counts

Counts

b3-H2O b6 y8
1.6 0
b4 ×10 2
1.2 y7
b5
Counts

y12 y13 y15 1

0.8
×10 1
0.4
Counts

0
200 600 1,000 1,400 1,800 2,200 2,600 3,000 6.7 6.8 6.9 7.0 7.1 7.2 7.3 7.4
Acquisition time (min)
Mass-to-charge (m/z)

Figure 2. MS/MS spectrum and the optimized MRM chromatography for peptide SSTVPIPTVNQYLYFLFAPTLIYR.

3
Glycopeptide quantification
An 1290 Infinity UHPLC system coupled
with Agilent Jet Stream Technology, and
a 6495B Triple Quadrupole LC/MS system
with a mass range of 3,000 u were used
for glycopeptide quantitation with the
following results (Figure 3): ×10 2 A B
Blank

Counts
2.48 Amount of total mAb %RSD
• Excellent precision and accuracy digest on-column (ng) (n = 10) % Accuracy
at all levels including injection 2.46
0.0197 18.5 97.5
×10 2
amount as low as 0.0197 ng 0.0394 18.3 104.2
0.0098 ng
Counts
2.48
(130 amol) of total mAb digest 0.0591 12.5 108.3
2.46
on-column (18.5 % RSD, 97.5 % 0.0985 9.3 102.4
×10 2
accuracy). This target peptide is 0.197 13.0 95.6
0.0197 ng
Counts

estimated to represent about 10 % 2.5 0.394 5.9 84.8

of the total protein so LOQ for this 0.985 5.7 102.1
glycopeptide is estimated at about ×10 2
1.97 1.6 105.5
0.0394 ng
13 amol on-column
Counts

2.6 19.7 2.9 99.7

• Reproducible responses for ×10 2

product ions with extended mass 0.0591 ng
Counts

range (m/z 204.1 ~ m/z 2,227.9) 2.6

0.90 0.95 1.00 1.05 1.10

• Excellent retention time (RT) Acquisition time (min)
reproducibility (RSD% = 0.47 % for
C
n = 90) 932.7 & 204.1
20,000
18,000 932.7 & 2,227.9 1,400
• Excellent linearity at low injection 16,000 932.7 & 2,065.8 1,200
levels with small fold changes 14,000 932.7 & 1,392.6
Response

Response
1,000
12,000
(0.0197 ng ~ 19.7 ng, R2 = 0.998) 10,000
932.7 & 366.1 800
8,000 600
6,000 400
4,000
2,000 Zoom 200
0 0
1 2 3 4 5 6 7 8 9 10 1 2 3 4 5 6 7 8 9 10
Replicate injection Replicate injection

×10 4
2 D
1.9 EEQYN(+1606.586706)STYR
1.8 y = 943.671754*x – 5.093398
1.7 R2 = 0.99814103
1.6
1.5
1.4
1.3
1.2
1.1 ×10 2
Response

1.0
0.9 3.0
0.8 2.6
0.7 2.2
Response

0.6 1.8
0.5 1.4
0.4 1.0
0.3 0.6
0.2 Zoom 0.2
0.1 -0.2
0 0 0.04 0.08 0.12 0.16 0.20 0.24 0.28
-0.1

-1 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
Concentration (ng of mAb digest on-column)

Figure 3. Quantification for G1F glycopeptide EEQYN[+1606.6]STYR.

4
 HeLa membrane peptide • The quantifier response for each of • Good linearity was observed
quantification these three peptides were plotted (R2 ranges from 0.989 to 0.997).
Three peptides from the HeLa against the relative injection
amount on‑column (left plots) as • Reproducible responses for
membrane‑enriched sample digest were
quantified following the same protocol as the stoichiometry of these peptides product ions with extended mass
the glycopeptide to test high m/z product were unknown in the sample. range (up to m/z 2429.3) were
ion behavior (Figure 4). observed (right plots).

×10 4
YFAGNLASGGAAGATSLCFVYPLDFAR
1.0 y = 202.884088*x – 54.476396 12,000
0.9 R 2 = 0.99576872 1,398.7 & 718.4
0.8 10,000 1,398.7 & 2,060.0
0.7 1,398.7 & 1,915.9
Response

8,000
Response

0.6 1,398.7 & 881.5

0.5 6,000 1,398.7 & 175.1
0.4
0.3 4,000
0.2 2,000
0.1
0 0
-0.1 1 2 3 4 5
0 4 8 12 16 20 24 28 32 36 40 44 48 52 Replicate injection
Concentration (relative amount on-column)
×10 4
1.1 NLIPFDQMTIEDLNEAFPETK 12,000 1,398.7 & 718.4
1.0 y = 205.558590*x – 13.485689
R 2 = 0.99704134 1,398.7 & 2,060.0
0.9 10,000
1,398.7 & 1,915.9
0.8
Response

8,000 1,398.7 & 881.5

0.7
Response

0.6 6,000 1,398.7 & 175.1

0.5
0.4 4,000
0.3 2,000
0.2
0.1 0
0 1 2 3 4 5
-0.1 Replicate injection
0 4 8 12 16 20 24 28 32 36 40 44 48 52
Concentration (relative amount on-column)
×10 3
1.5 SSTVPIPTVNQYLYFLFAPTLIYR
1.4 y = 29.206852*x – 10.518556 1,6000 1,398.7 & 718.4
1.3 R 2 = 0.98908869
1.2 1,4000 1,398.7 & 2,060.0
1.1 1,2000 1,398.7 & 1,915.9
1.0
Response

0.9 1,0000 1,398.7 & 881.5

Response

0.8 1,398.7 & 175.1

0.7 8000
0.6
0.5 6000
0.4 4000
0.3
0.2 2000
0.1
0 0
-0.1 1 2 3 4 5
0 4 8 12 16 20 24 28 32 36 40 44 48 52 Replicate injection
Concentration (relative amount on-column)

Figure 4. Quantification for peptides from a HeLa membrane digest.

5
Conclusions
The Agilent 6495B Triple Quadrupole
LC/MS system has a mass range of
3,000 u, which is useful for detecting
high m/z peptide ions. One benefit of
using high m/z precursor or product
ions, particularly in a complex digest, is
these ions have less background noise
compared to those with low m/z. Some
high m/z product ions may provide
important biological information such as
location and size of post-translational
modifications, for example glycosylation.

This application notes demonstrates

the excellent reproducibility for MRM
transitions with high m/z product ions
achieved on the Agilent 6495B Triple
Quadrupole LC/MS system. The work
shown here uses Agilent Jet Stream
Technology at standard flow rates and
resulted in outstanding precision and
accuracy for both glycopeptide and HeLa
membrane peptide quantification.

www.agilent.com
For Research Use Only. Not for use in diagnostic
procedures.

This information is subject to change without notice.

© Agilent Technologies, Inc., 2016

Published in the USA, November 30, 2016
5991-7298EN